Dextran-shelled oxygen-loaded nanodroplets modulate macrophages killing and inflammatory response to Enterococcus faecalis.

Chronic wounds are associated with inflammation, infections, and hypoxic environment. Macrophages play a crucial role in wound healing removing bacteria and secreting signal molecules to coordinate tissue repair. Recently, dextran-shelled Oxygen-Loaded NanoDroplets (OLNDs) have been proposed as new tools to counteract hypoxia in chronic wounds. Here we investigated the effects of OLNDs on Enterococcus faecalis (E. faecalis) killing and the secretion of inflammatory and angiogenic factors by murine (BMDM) and human (dTHP-1, differentiated THP-1) macrophages, in normoxia and hypoxia. Both OLNDs and Oxygen-Free NanoDroplets (OFNDs) significantly increased reactive oxygen species production by BMDM in normoxia (4.1 and 4 fold increase by 10% OLNDs and OFNDs, respectively, after 120 min) and hypoxia (3.8 and 4 fold increase by 10% OLNDs and OFNDs respectively) but not by dTHP-1. Moreover, only OLNDs induced nitric oxide secretion by BMDM in normoxia. Consequently, both nanodroplets improved E. faecalis killing by BMDM in normoxia (% of killing OLNDs = 44.2%; p < 0.01; OFNDs = 41.4%; p < 0.05) and hypoxia (% of killing OLNDs = 43.1%; p < 0.01; OFNDs = 37.7%; p < 0.05), while dTHP-1-mediated killing was not affected. The secretion of the inflammatory cytokines (TNFα, IL-6, IL-1ß) induced by E. faecalis infection in dTHP-1 was reduced by both types of nanodroplets, suggesting a novel anti-inflammatory activity of the dextran shell. Instead, the increase of VEGF induced by hypoxia was reduced only by OLNDs. These data provide new knowledge on the effects of OLNDs as innovative adjuvant in chronic wounds healing promoting bacterial killing and reducing inflammation.

In Vitro Study Evaluating the Effect of Different Immunosuppressive Agents on Human Polyomavirus BK Replication.

BACKGROUND: Human polyomavirus BK (BKPyV) is the etiologic agent of polyomavirus-associated nephropathy, a leading cause of kidney transplant dysfunction. Because of the lack of antiviral therapies, immunosuppression minimization is the recommended treatment. This strategy offers suboptimal outcomes and entails a significant risk of rejection. Our aim was to evaluate the effect of different immunosuppressive drugs (leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus) and their combinations in an in vitro model of BKPyV infection. METHODS: Human renal tubular epithelial cells were infected with BKPyV and treated with leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus, administered alone or in some combination thereof. Viral replication was assessed every 24 hours (up to 72 hours) by BKPyV-specific quantitative real-time polymerized chain reaction for the VIRAL PROTEIN 1 sequence in cell supernatants and by western blot analysis targeting the viral protein 1 and the glyceraldehyde 3-phosphate dehydrogenase on total protein lysates. Results were described as viral copies/mL and compared between treatments at any prespecified time point of the study. RESULTS: The highest inhibitory effects were observed using leflunomide or everolimus plus mycophenolic acid (mean BKPyV replication log reduction 0.28). The antiviral effect of everolimus persisted when it was used in combination with tacrolimus (mean BKPyV replication log reduction 0.27). CONCLUSIONS: Our experience confirms that everolimus has anti-BKPyV properties and prompts future research to investigate possible mechanisms of action. It also provides a rational basis for targeted clinical trials evaluating alternative immunosuppressive modification strategies.

Viral Genomic Characterization and Replication Pattern of Human Polyomaviruses in Kidney Transplant Recipients.

Human Polyomavirus (HPyV) infections are common, ranging from 60% to 100%. In kidney transplant (KTx) recipients, HPyVs have been associated with allograft nephropathy, progressive multifocal leukoencephalopathy, and skin cancer. Whether such complications are caused by viral reactivation or primary infection transmitted by the donor remains debated. This study aimed to investigate the replication pattern and genomic characterization of BK Polyomavirus (BKPyV), JC Polyomavirus (JCPyV), and Merkel Cell Polyomavirus (MCPyV) infections in KTx. Urine samples from 57 KTx donor/recipient pairs were collected immediately before organ retrieval/transplant and periodically up to post-operative day 540. Specimens were tested for the presence of BKPyV, JCPyV, and MCPyV genome by virus-specific Real-Time PCR and molecularly characterized. HPyVs genome was detected in 49.1% of donors and 77.2% of recipients. Sequences analysis revealed the archetypal strain for JCPyV, TU and Dunlop strains for BKPyV, and IIa-2 strain for MCPyV. VP1 genotyping showed a high frequency for JCPyV genotype 1 and BKPyV genotype I. Our experience demonstrates that after KTx, HPyVs genome remains stable over time with no emergence of quasi-species. HPyVs strains isolated in donor/recipient pairs are mostly identical, suggesting that viruses detected in the recipient may be transmitted by the allograft.

Particulate matter exposure increases JC polyomavirus replication in the human host.

BACKGROUND: Human polyomaviruses (HPyVs) asymptomatically infect the human population during childhood and establish latency in the host. Viral reactivation and urinary excretion can occur when the immune system is impaired. Exposure to particulate air pollution, including the PM10/PM2.5 components, is a public health problem and has been linked to several disorders. Studies assessing the relationship between PM10/PM2.5 exposure and viral replication are lacking. OBJECTIVES: To investigate the relationship between HPyVs viruria and PM10/PM2.5 exposures. METHODS: Individual environmental exposure was assessed in 50 healthy adult volunteers using a chemical transport model (CTM) with a municipality resolution for daily PM10 and monitoring stations data for daily PM2.5 exposures. For each subject, a urine sample was collected, and HPyVs (JCPyV, BKPyV, MCPyV, HPyV6, HPyV7 and HPyV9) loads were determined. Zero-inflated negative binomial (ZINB) regression was used to model the count data, as it contained excessive zeros. Covariates were chosen by stepwise selection. RESULTS: HPyVs DNA was detected in 54% (median:87.6*105 copies/ml) of the urine samples. JCPyV was the prevalent (48%, (median viral load:126*105 copies/ml). Considering the load of the most frequently measured HPyVs, JCPyV, in the count-part of the ZINB model, every unitary in PM measured 2 days before urine collection (PM Day -2) was associated with an increase in JCPyV load (PM10: +4.0%, p-value = 0.002; PM2.5: +3.6%, p-value = 0.005). In the zero-part, the significant predictor was the PM10 measured 5 days before urine collection (+3%, p-value = 0.03). CONCLUSIONS: The environmental levels of PM10/PM2.5 increase the JCPyV viruria. Our findings emphasize the need for studies assessing the influence of air pollution exposure on the risk of viral reactivation.

Multiplex array analysis of circulating cytokines and chemokines in natalizumab-treated patients with multiple sclerosis.

Natalizumab greatly reduces inflammatory relapses in multiple sclerosis (MS) by blocking the integrin-mediated leukocyte traffic to the brain, but less is known about its effects on the systemic immunity. We measured 48 cytokines/chemokines in sera from 19 natalizumab-treated MS patients. Serum concentrations of both anti-(IL-10, IL1ra) and pro-inflammatory (IL7, IL16) molecules decreased after 21-month treatment, without associations to unbalanced Th2/Th1cytokine ratios, clinical responses, and blood/urine replication of polyomavirus JC (JCPyV). No patient developed the JCPyV-related progressive multifocal leukoencephalopathy (PML), the major risk factor of natalizumab therapy. Our data suggest that natalizumab has marginal impact on the systemic immunity.

High frequency of Merkel cell polyomavirus DNA in the urine of kidney transplant recipients and healthy controls.

BACKGROUND: Polyomavirus (PyV) infection is common, ranging from 60% to 100% depending on the virus. The urinary excretion rates of JC virus (JCV) and BK virus (BKV) have been extensively studied, but less is known about the more recently discovered PyVs. OBJECTIVES: To investigate the urinary excretion of Merkel cell PyV (MCPyV), which is associated with Merkel cell carcinoma (MCC), in kidney transplant recipients and healthy subjects, as well as those of lymphotropic polyomavirus (LPV), which was isolated from the B-cells of African green monkeys but has also been found in human blood, JCV and BKV. STUDY DESIGN: Urine samples were collected from 62 adult (ATP) and 46 pediatric (PTP) kidney transplant recipients and from 67 adult (AHC) and 40 pediatric (PHC) healthy controls. DNA was isolated and analyzed using real-time PCR (Q-PCR) to determine the viral loads of MCPyV, LPV, JCV and BKV. RESULTS: MCPyV DNA was more frequently detected (p<0.05) in the PTP (36.9%) and PHC (30.0%) groups compared to JCV (8.7% in PTP, 12.5% in PHC), BKV (6.5% in PTP, 2.5% in PHC), and LPV (2.2% in PTP, 5% in PHC) and in the AHC (47.8%) group compared to BKV (13.4%) and LPV (0%). CONCLUSIONS: Based on the results, it could be concluded that: (a) Despite the rarity of MCC, MCPyV is a common infection; (b) MCPyV demonstrates an excretion pattern similar to those of JCV and BKV, persisting in the kidney and infecting skin cells upon reactivation, with subsequent integration and transformation.